Treatment of viral infections

ABSTRACT

Treatment of animals or humans carrying or infected with HTLV-I, HTLV-I (including HTLV-I-associated leukemias or lymphomas), non-A, non-B hepatitis virus, hepatitis B virus, EBV, equine infectious anaemia or other lentiviruses or having antibodies to said viruses is disclosed using the compound 3&#39;-azido-3&#39;-deoxythymidine or a pharmaceutically acceptable basic salt thereof. Also disclosed is the use of the 5&#39;-mono-, di- and triphosphate of 3&#39;-azido-3&#39;-deoxythymidine or a pharmaceutically acceptable basic salt thereof for the same purpose.

This is a continuation of application Ser. No. 07/792,812 filed on Nov.15, 1991, this is a continuation of application Ser. No. 07/670,499filed on Mar. 15, 1991, now abandoned, which is a continuation of Ser.No. 07/288,735 filed Apr. 29, 1988, now abandoned which is acontinuation of Ser. No. 06/839,795 filed Mar. 14, 1986, now abandoned.

This invention is directed to the treatment of humans infected withHTLV-I and HTLV-II, including HTLV-I-associated leukemias and lymphomas,non-A, non-B hepatitis virus, hepatitis B virus, and Epstein-Barr virus(EBV), as well as to the treatment of animals infected with equineinfectious anaemia and other lentiviruses.

It has now been discovered that the compound known as3'-azido-3'-deoxythymidine having the formula I ##STR1## is useful intreating humans identified as having HTLV-I and HTLV-II infection,including HTLV-I-associated leukemias or lymphomas, non-A, non-Bhepatitis, hepatitis B, or EBV infections. It is also useful in treatinganimals identified as having equine infectious anaemia or otherlentivirus infections.

The compound of formula I may be administered per se or in the form of apharmaceutically acceptable salt to the infected animal or human, e.g.,an alkali metal salt such as sodium or potassium salts, an alkalineearth metal salt or an ammonium salt such as tetraalkylammonium salts(all of which are hereinafter referred to as a pharmaceuticallyacceptable base salt).

The salts of the compound of formula I convert to the compound per seafter being administered. The compound 3'-azido-3'-deoxythymidinepenetrates into the virally infected cells after contacting same and isconverted by cellular enzymes to the monophosphate thereof. Themonophosphate is then converted by the cellular enzymes to thediphosphate of the compound of formula I and ultimately to thetriphosphate of the compound of formula I.

The triphosphate of the compound of formula I acts after contact withthe virus to prevent replication of the virus.

Thus the compound of formula I can be said to be a pro-drug afterentering the animal or human cells since it is an intermediate(precursor) to the 5'-mono-, di- and triphosphate thereof.

It is believed that the 5'-mono-, di- and triphosphates of the compoundof formula I can also be said to be prodrugs since they would invariably(at least in part) be hydrolized in the animal or human to3'-azido-3'-deoxythymidine which is taken up by the cells.

As another feature of this invention, there is also disclosed the methodof administering to an animal or human in need thereof the 5'-mono-, di,or triphosphate of the compound of formula I or their pharmaceuticallyacceptable base salts (i.e., alkali metal, alkaline earth or ammoniumsalt) to treat the above-mentioned virus infections and to inhibit thereplication of the virus in infected animal or human cells. The5'-mono-, di- and triphosphates of 3'-azido-3'-deoxythymidine are of theformulas II, III and IV respectively. ##STR2##

The present invention thus discloses compounds of formulas I, II, IIIand IV and their appropriate salts for use in the treatment of theconditions referred to above, as well as the use of such compounds inthe preparation of pharmaceutical formulations for the treatment of suchconditions. The above mentioned pharmaceutically acceptable salts may beprepared in a conventional manner, e.g., treatment of the compound withan appropriate base.

In general for the treatment of the above mentioned virus infections, asuitable effective dose of the 3'-azido-3'-deoxythymidine, itspharmaceutically acceptable basic salts, its mono-, di- or triphosphatesor their pharmaceutically acceptable basic salts (all of which arehereinafter referred to as the administered ingredient) will be in therange 10 to 250 mg per kilogram body weight per day, preferably in therange of 25 to 100 mg per kilogram body weight per day and mostpreferably in the range 30 to 60 mg per kilogram body weight per day.The desired dose is preferably presented as two, three, four or moresub-doses administered at appropriate intervals throughout the day.These sub-doses may be administered at unit dosage forms, for example,containing 5 to 500 mg, preferably 10 to 200 mg and most preferably 20to 100 mg of active ingredient per unit dosage form.

Administration may be by any suitable route including oral, rectal,nasal, topical (including buccal and sublingual), vaginal and parenteral(including subcutaneous, intramuscular, intravenous and intradermal)with oral or parenteral being preferred. It will be appreciated that thepreferred route may vary with, for example, the condition and age of therecipient.

A preferred dose is administered to achieve peak plasma concentrationsof the compound of formula I, its mono-, di- or triphosphate or itspharmaceutically acceptable salts of from about 1 to about 100 μM,preferably about 2 to 80 μm, most preferably about 3 to about 50 μm.This may be achieved, for example, by the intravenous injection of asterile 0.1 to 5% solution of the administered ingredients in saline asa bolus containing about 1 to about 20 mg/kg of the active ingredient.Desirable blood levels may be maintained by a continuous infusion toprovide about 0.03 to about 1.0 mg/kg/hour or by intermittent infusionsproviding about 0.12 to about 30 mg/kg of the administered ingredient.

The administered ingredients may be used in therapy in conjunction withother medicaments such as9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine,9-(2-hydroxyethoxymethyl)guanine (acyclovir),2-amino-9-(2-hydroxyethoxymethyl)purine, suramin, ribavirin,antimoniotungstate (HPA-23), interferon, e.g., α interferon, interleukinII, and phosphonoformate (Foscarnet) or in conjunction with other immunemodulation including bone marrow or lymphocyte transplants ormedications such as levamisol or thymosin which would increaselymphocyte numbers and/or function as is appropriate.

The compound of formula I, 3'-azido-3'-deoxythymidine, (sometimes alsoreferred as azidothymidine) is disclosed in J. R. Horwitz et al, J. Org.Chem., 29, Jul. 1964, pp. 2076-2078; M. Imazawa et al, J. Org. Chem.,43(15) 1978, pp. 3044-3048; also see Biochemical Pharmacology, Vol. 29,pp. 1849-1851; C. J. Kreig et al, Experimental Cell Research 116 (1978)pp. 21-29. Also see W. Ostertag et al, Proc. Nat. Acad. Sci. USA 71(1974) for some of its biological activities.

While it is possible for the administered ingredients to be administeredalone, it is preferable to present them as part of a pharmaceuticalformulation. The formulations of the present invention comprise at leastone administered ingredient, as above defined, together with one or moreacceptable carriers thereof and optionally other therapeuticingredients. The carrier(s) must be "acceptable" in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

The formulations include those suitable for oral, rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal)administration. The formulations may conveniently be presented in unitdosage form, e.g., tablets, sustained release capsules, and may beprepared by any methods well known in the art of pharmacy.

Such methods include the step of bringing into association the to beadministered ingredients with the carrier which constitutes one or moreaccessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both and then,if necessary, shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Formulations suitable for topical administration in the mouth includelozenges comprising the ingredients in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the ingredient to be administeredin a suitable liquid carrier.

Formulations suitable for topical administration to the skin may bepresented as ointments, creams, gels and pastes comprising theingredient to be administered and a pharmaceutically acceptable carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for nasal administration wherein the carrier is asolid include a coarse powder having a particle size, for example, inthe range 20 to 500 microns which is administered in the manner in whichsnuff is taken, i.e., by rapid inhalation through the nasal passage froma container of the powder held close up to the nose. Suitableformulations wherein the carrier is a liquid, for administration, as forexample, a nasal spray or as nasal drops, include aqueous or oilysolutions of the active ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended animal or human; and aqueous andnon-aqueous sterile suspensions which may include suspending agents andthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude flavouring agents.

The LD₅₀ for 3'-azido-3'-deoxythymidine in mice and rats was found to begreater than 750 mg/kg.

The following examples illustrate the invention.

EXAMPLE 1 Preparation of 2,3'-Anhydrothymidine

Thymidine (85.4 g; 0.353 mol) was dissolved in 500 mL dry DMF (dimethylformamide) and added to N-(2-chloro-1,1,2-trifluoroethyl)diethylamine(100.3 g; 0.529 mol) [prepared according to the method of D. E. Ayer,μmed. Chem. 6, 608 (1963)]. This solution was heated at 70° C. for 30minutes then poured into 950 mL ethanol with vigorous stirring. Theproduct precipitated from this solution and was filtered. The ethanolsupernatant was refrigerated then filtered to yield a total of 47.75 g(0.213 mol; 60.3%) of 2,3'-anhydrothymidine, mp=228°-230° C.

EXAMPLE 2 Preparation of 3'-Azido-3'-deoxythymidine

2,3'-Anhydrothymidine (25 g; 0.1115 mol) and NaN₃ (29 g; 0.446 mol) wassuspended in a mixture of 250 mL DMF and 38 mL H₂ O. The reaction wasrefluxed for 5 hours at which time it was poured into 1 liter of H₂ O.This aqueous solution was extracted with ethyl acetate (EtOAc) (3×700ml). The EtOAc was dried over Na₂ SO₄, filtered, and then EtOAc wasremoved in vacuo to yield a viscous oil. This oil was stirred with 200mL water resulting in a solid, 3'-azido-3'-deoxythymidine, 9.15 g(0.0342 mol; 30.7%) mp=116°-118° C.

EXAMPLE 3 Preparation of Sodium Salt of 3'-Azido-3'-deoxythymidine

Approximately one gram of 3'-azido-3'-deoxythymidine was dissolved in 50mL of distilled water. The pH was adjusted to 12 with 1N NaOH.Approximately half of the solution was freeze dried. A white powder, thesodium salt of 3'-azido-3'-deoxythymidine as the 0.6 hydrate, 0.415 g,resulted.

Analysis calculated for C₁₀ H₁₂ N₅ NaO₄. 6/10 H₂ O. Calculated: C,40.03;H,4.43; N,23.34; Na,7.66 Found: C,39.88; H,4.34; N,23.29; Na,7.90

EXAMPLE 4 Preparation of 5'-Monophosphate of 3'-Azido-3'-deoxythymidine

3'-Azido-3'-deoxythymidine (0.5 g, 1.87 mmol) was dissolved in 5 mL oftriethyl phosphate and the mixture was cooled to -5° C. Phosphorusoxychloride (0.685 mL, 7 mmol) was added in one portion to the rapidlystirred solution which was then maintained at -10° C. for 22 hours. Analiquot was removed and added to concentrated ammonium hydroxide.Analysis of this sample on TLC (cellulose, n-PrOH:H2O, 7:3 v/v) showedno remaining starting material and a single fluorescent spot with lowermobility than the nucleoside. The reaction mixture was poured onto 20 mLof ice and water. This was placed in an ice bath and the pH of thesolution was adjusted to a value of 7.5 by the addition of 2N NaOH. Thebasic mixture was extracted once with chloroform and once with ether.The aqueous layer was again adjusted to give a pH of 7.5 andconcentrated in vacuo remove residual organic solvent. The material wasstored at -10° C. until purified as follows:

Deactivated charcoal was prepared by washing coconut charcoal (50-200mesh, 100 g) with 500 mL of 1N HCl, 3 L of water, 35 mL of 3% toluene in95% ethanol, 600 mL of 95% ethanol and finally extensively with water.Deactivated charcoal (12 mL of settled wet charcoal) was added withstirring to the monophosphate solution (0.72 g, 1.8 mmol, 30 mL). Thesupernatant was decanted and the charcoal was washed with 150 mL ofwater. The nucleotide was eluted from the charcoal by washing with 120mL of 1.5M ammonium hydroxide in 50% ethanol. This solution was filteredthrough a 0.22 micron filter, concentrated in vacuo to 10 mL, filteredthrough a Amicon Centriflo CF-25 membrane, and lyophilized. The yield ofdiammonium 3'-azido-3'-deoxythymidine-5'-monophosphate was 0.36 g (0.94mmol, 52%). This compound was characterized as a nucleoside5'-monophosphate by the ability of 5'-nucleotidase to degrade it to thenucleoside.

EXAMPLE 5 Preparation of the 5'-Di- and Triphosphate of3'-Azido-3'-deoxythymidine

The di- and triphosphate of 3'-azido-3'-deoxythymidine were preparedfrom the ammonium salt of the 5'-monophosphate of3'-azido-3'-deoxythymidine by a four step sequence.

Step I--Preparation of Bis-(Tri-n-butylammonium) Pyrophosphate

A column of DOW 50 pyridinium resin was prepared by pouring 40 mL ofresin into a 25 cm diameter column and washing with water until no morecolor eluted. Sodium pyrophosphate decahydrate (1.12 g, 2.51 mol) wasdissolved in 30 mL of water and applied to the column. The column waseluted with water. A 125 mL fraction of the eluant which contained UVabsorbing material was collected. The volume was reduced to 10 mL invacuo and tri-n-butyl amine (1.2 mL) was added. The volume was reducedin vacuo and the residue was dried by coevaporation with pyridine fourtimes. The product was stored in a freezer (-5° C.).

Step II--Preparation of the Acid Form of the 5'-Monophosphate of3'-Azido-3-deoxythymidine

The acid form of the monophosphate was prepared by passing the ammoniumsalt (0.1 g, 0.283 mmol) dissolved in 6 mL of water, through a 1.5 mL(10 eq.) column of DOW 50 H+.

Step III--Preparation of Phosphoromoropholidate Derivative

The hydrogen form of the monophosphate, 0.283 mMol, was dissolved in 9mL of water. Morpholine (99 UL, 1.13 mmol, 4 eq.) was added and thesolution heated to reflux. Dicyclohexyl carbodiimide (0.234 g, 1.13 mmol, 4 eq.) dissolved in t-butanol (5 mL) was added over a three-hourperiod. The reaction was refluxed overnight. The reaction was cooled toroom temperature, filtered, and the solvents removed in vacuo. Ethanolwas added and evaporated in vacuo four times. The residue was dissolvedin methanol and the phosphoromorpholidate precipitated by the additionof ether. The precipitate was triturated with ether four times and driedon a rotary evaporator. A yield of 97 mg, 50%, was obtained.

Step IV--Preparation of the 5'-Di- and Triphosphates of3'-Azido-3'-deoxymidine

The phosphoromorpholidate derivative was dried by removal of pyridine invacuo four times. The bis-(tri-n-butylammonium) pyrophosphate was alsodried by removal of pyridine in vacuo. The phosphoromorpholidate wasdissolved in pyridine, 5 mL, and added to the vessel containing thepyrophosphate reagent. The reaction was allowed to continue overnite atroom temperature. The pyridine was removed in vacuo. Water was added tothe residue and removed in vacuo three times. The residue was frozen.

The reaction mixture (0.09 g) was thawed and dissolved in 50 mL ofwater. The solution was applied to a column (1×10 cm) of DEAE SephadexA-25 which had been equilibrated with 50 mM ammonium bicarbonate. Thephosphates were eluted with a 300 mL linear gradient of 50-800 mMammonium bicarbonate. The fractions containing the diphosphatenucleotide were pooled as were those containing the triphosphatenucleotide. The pooled diphosphate and triphosphate fractions were eachdried in vacuo, redissolved in water, dried again, redissolved in waterand and lyophilized. The yields were: the diphosphate as the triammoniumsalt, 0.014 g; the triphosphate, as the tetrammonium salt, 0.002 g.

EXAMPLE 6 Enzymatic Synthesis of the 5'-Triphosphate of3'-Azido-3'-deoxythymidine

The 5'-triphosphate was synthesized from the 5'-diphosphate usingpyruvate kinase and nucleoside diphosphate kinase. The reaction mixturecontained: 6 mM 3'-azido TDP, 12 mM adenosine triphosphate, 40 mM MgCl2,40 mM potassium piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPESbuffer, pH 6.8), 30 mM phosphoenolpyruvate, 40 IU/ml nucleosidediphosphate kinase and 100 IU/ml pyruvate kinase in a final volume of 5mL. The reaction mixture was incubated at 37° C. for 5 days. Thereaction mixture was applied to a column (2.5×10 cm) of DEAE SephadexA-25 which had been equilibrated with ammonium bicarbonate. Thenucleotides were eluted with a gradient of 100-1000 mM ammoniumbicarbonate. Fractions containing the triphosphate were pooled, andevaporated to dryness in vacuo. The compound was further purified usinga preparative HPLC column (Whatman, Inc., Magnum 9 SAX) eluted with agradient of 10-1000 mM potassium phosphate, ph 3.5. The resultingcompound was further purified using a DEAE Sephadex A-25 column asabove. The fractions containing the tetrammonium3'-azido-3'-deoxythymidine-5'-triphosphate were pooled, dried in vacuo,redissolved in water and lyophilized to yield 0.008 g (0.01 mmol).

EXAMPLE 7

    ______________________________________                                        Tablet Formulation                                                            ______________________________________                                        3'-Azido-3'-deoxythymidine                                                                             100 mg                                               Lactose                  200 mg                                               Starch                   50 mg                                                Polyvinylpyrrolidone     5 mg                                                 Magnesium stearate       4 mg                                                                          359 mg                                               ______________________________________                                    

Tablets were prepared from the foregoing ingredients by wet granulationfollowed by compression.

EXAMPLE 8 Capsule

A two part soft gelatin is prepared by placing the tablet formulation ofExample 7 in a two part capsule.

EXAMPLE 9

    ______________________________________                                        Sterile Injectable Formulation                                                ______________________________________                                        3'-Azido-3"-deoxythymidine 0.125  g                                           Sterile, pyrogen-free, pH 7 phosphate buffer, q.s. to                                                    25     ml                                          ______________________________________                                    

The formulation is sterilized by heat and then placed in a sealedcontainer, e.g., glass, so that it may be administered by infusion or byinjection.

EXAMPLE 10

The acid forms of the 5'-mono-, di- and triphosphate nucleotides of3'-azido-3'-deoxythymidine are prepared by passing the correspondingammonium salts through DOW 50 H+ columns and lyophilizing the solutionsobtained thereby.

We claim:
 1. A method of treating an HTLV-I infection in a host in need thereof comprising administering to said host an effective treatment amount of 3'-azido-3'-deoxythymidine or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1, in which the compound or salt is administered orally or parenterally.
 3. The method of claim 1, in which the compound or salt is administered orally or parenterally.
 4. A method of treating an HTLV-II infection in a host in need thereof comprising administering to said host an effective HTLV-II treatment amount of 3'-azido-3'-deoxythymidine or a pharmaceutically acceptable salt thereof. 